CN1270034A - application of coenzyme in histocyte chemistry and prevention and cure of radiation injury - Google Patents

Abstract

The application of coenzyme NADH in preventing and curing the chemical and radiation injury of histocyte is disclosed. Said coenzyme can selectively and early prevent the normal histocyte from being damaged by chemical and radiation, DNA damage and death generation. Said NADH can block the expression of death moleculae Caspase-3 and Caspase-8, inhibit the release of cytochrome C from chondriosome to kytoplasm, suppress the lowering down of membrane potential of chondriosome and the raising up of free calcium ions, reduce the ROS release, and promote growth of normal cells. Externally supplementing NADH can repair damaged histooyte and suppress death. It can be used for chemicotherapy and ratiotherapy of cancers and preventing and treating the damage from chemical and nuclear weapons.

Description

Application of coenzymes in histocytochemistry and radiation damage prevention.

The invention discloses the application of coenzyme in the prevention and treatment of histocytochemistry and radiation damage. The coenzyme NADH can be used as a protective agent for radiotherapy and chemotherapy, improve the treatment intensity of radiotherapy and chemotherapy, improve the quality of life of patients, and can be used for the prevention and treatment of chemical and nuclear weapon damage under the conditions of modern warfare. effective biological warfare agent.

Currently known, anticancer drugs, radiation and chemical warfare agents are toxic to normal bone marrow, kidney, liver, nervous system and mucosal epithelium. These toxicities limit the therapeutic intensity of anticancer drugs and radiotherapy, significantly affecting the quality of life and threatening life. Approaches to improve treatment include increasing the sensitivity of malignant cells to chemotherapy or reducing the toxicity of chemotherapy drugs to normal tissues, and developing drugs that protect normal tissues (but not malignant cells) from damage. Due to the non-specificity of anti-tumor drugs to target cells, normal tissue cells are also killed. Therefore, how to avoid damage to normal cells during chemotherapy and clarify the mechanism involved in repairing DNA damage has important clinical significance. Nuclear, biochemical and new concept weapons add more difficulty to health and disease prevention. Therefore, non-combat attrition must be reduced. In recent years, research at home and abroad has shown that radiation, biological and chemical toxins can cause DNA damage in cells and induce normal human health. Apoptosis occurs in cells, causing disorder and destruction of normal physiological functions of the body. The pathological mechanism of many modern war injuries involves abnormal cell apoptosis/death process, and the induced ischemia, hypoxia, poisoning and oxidative stress can lead to apoptosis and necrosis of heart, brain, liver and other tissue cells. And then cause a variety of complications, based on the above-mentioned mechanism of mitochondrial apoptosis, people are looking for strategies to effectively control cell apoptosis/death from a new perspective, such as Ca ²⁺ chelating agents, free radical scavengers and cyclosporine A, etc. can inhibit mitochondrial permeability transition and inhibit cell death. For the prevention of acute radiation sickness in foreign countries, chemical drugs, Chinese herbal medicines, and biological agents are mainly used. The anti-radiation mechanism is also different, such as protecting the sulfhydryl group of target cell molecules, scavenging free radicals, and stabilizing DNA structures. Although in vitro experiments and animal Model studies have found a large number of effective radioprotectants, but they are rarely used in humans due to drug toxicity and other reasons. In recent years, the recombination of cytokines (such as GM-CSF) has successfully provided assistance for anti-radiation therapy, but due to its high price, it is impossible to become a preventive drug and widely used in the army. Therefore, the development of new cell drugs with obvious anti-toxic and anti-radiation effects, enhanced cell stress response, improved energy metabolism and enhanced resistance to and repair of DNA damage is essential for the prevention of nuclear, biological and chemical weapons, high temperature, trauma, etc. in the treatment of modern war wounds. The downside has far-reaching implications. NADH is a necessary coenzyme for cellular energy metabolism, and plays an important role in initiating the redox reaction of organisms and regulating the expression of cell membrane glycoprotein receptors. Our previous studies have shown that NADH can increase the synthesis of endogenous dopamine and improve the symptoms of Parkinson's syndrome. It is reported abroad that NADH can be used in the adjuvant treatment of Parkinson's syndrome, Alzheimer's disease and vomiting syndrome. At present, there are no research reports on the resistance of coenzyme NADH to chemical and radiation damage at home and abroad.

It is an object of the present invention to provide effective medicaments as a protection against chemical and radiation damage and against chemical and nuclear damage.

Our research shows that NADH can selectively prevent and early prevent normal tissue cells from chemical and radiation damage, and cell DNA damage is related to apoptosis. This enzyme can block the expression of apoptosis molecules Caspase-3 and Caspase-8, prevent the release of cytochrome C from mitochondria to cytoplasm, inhibit the decrease of mitochondrial membrane potential and the increase of free calcium ions during apoptosis, and reduce the release of ROS. Coenzyme NADH can promote normal cell proliferation and growth, and can inhibit apoptosis by up-regulating the expression of bcl-2 gene and down-regulating the expression of p53 gene. NADH protects the repair mechanism of cellular DNA damage by affecting the expression of apoptosis-related genes c-myc, c-erbB-2, c-fos, fas and cyclin. The concentration of NADH in normal tissue cells is significantly higher than that in tumor tissues, and the difference in NADH concentration between normal tissues and tumor tissues can be utilized to protect normal tissue cells from damage by radiotherapy and chemotherapy without affecting the best curative effect of radiotherapy and chemotherapy. The coenzyme can protect three lines of bone marrow hematopoietic stem cells, and can be used in combination with hematopoietic clone stimulating factors, which can stimulate hematopoiesis and protect hematopoietic stem cells from toxic damage caused by radiotherapy and chemotherapy, and is a protective factor for bone marrow. The enzyme can be used as a protective agent for chemical and radiation damage, improve the intensity of radiotherapy and chemotherapy, improve the quality of life of patients, and can be used as an effective biological warfare agent for preventing chemical and nuclear weapon damage under modern warfare conditions. It is characterized in that the effective concentration dissolved in normal saline is 20-1000ug/ml, and the effective ratio in excipients is 20-90%; The optimal ratio in the drug is 35-45%; the optimal action time is to give sufficient amount 12 to 24 hours before DNA damage occurs.

The present invention has the following advantages: 1. Discover a unique coenzyme NADH that protects normal tissue cells from chemical and radiation damage. 2. The anti-DNA damage effect of the coenzyme NADH provides a new drug for the treatment of modern war wounds. 3. The present invention clarifies the anti-apoptosis regulation mechanism of NADH from the gene and protein molecular levels, and provides a new theoretical and experimental basis for the reversal of apoptosis and DNA damage repair.

The following examples describe the experimental materials of Example 1 of the present invention in detail: 1. Human normal cell lines: PC12 neuron cell line, L02 liver cell line, SL-7 kidney cell line and MEF alveolar endothelial cell line are all in this room The cells were subcultured in a carbon dioxide incubator at 37°C and 5% CO ₂ saturated humidity by adding RPMI-1640 medium containing 10% inactivated fetal bovine serum, 1.2% glutamine, and 1% penicillin and streptomycin. 2. Main reagents: NADH, cisplatin, doxorubicin, rotenone, PARP polyclonal antibody, cytochrome C monoclonal antibody, anti-cyclin A monoclonal antibody 6E6, anti-cyclin B ₁ monoclonal antibody V152, anti-cyclin D ₁ monoclonal antibody DCS-6, anti-p53 protein monoclonal antibody D0-7, 3. anti-c-myc protein monoclonal antibody C-33, anti-bc1-2 protein monoclonal antibody 124, anti-c-erbB-2 protein monoclonal antibody NCL-CB11 , anti-c-fos protein monoclonal antibody UB-06341, fluorescent staining probes (PI, Rhodamin 123, Fluo-3-AM, Snalf-Calcein-1, H ₂ DCF, Hoechest 33342), Tunel apoptosis detection kit, RT -PCR reaction reagents. Experimental method: 1. MTT colorimetric assay 1.1 The effect of NADH on the proliferation activity of PC12 cells

The PC12 cells were subcultured in a 96-well plate at a density of 1.5-4.5×10 ³ /well, and cultured in a culture solution containing 50-600 μg/ml NADH for 48-72 hours before detection. 1.2 Cytotoxic effect of cisplatin on PC12 cells

PC12 cells were subcultured and inoculated in 96-well plates, and culture solutions containing cisplatin at a concentration of 0.075, 0.15, 0.3, and 0.6 mg/ml were added and incubated for 10 hours before detection. 1.3 NADH prevents the toxic effect of cisplatin on PC12 cells

After PC12 cells were cultured for 48 hours in culture solutions containing 400 μg/ml NADH and NADH-free, they were detected after adding culture solutions containing 0.01, 0.1 and 1 mM cisplatin for 10 hours. 1.4 NADH inhibits the toxic effect of cisplatin on PC12 cells

One group of PC12 cells was cultured for 10 hours by adding culture medium containing 400 μg/ml NADH and 0.01, 0.1, 1 mM cisplatin at the same time, and the other group was cultured by adding cisplatin-containing culture medium for PC12 cells for 10 hours before adding NADH After culturing for 48 hours, the cell proliferation activities of the two groups were compared. 2. Flow Cytometry Detection

After subculture, PC12 cells were divided into two groups. One group was added with 0.3 mg/ml cisplatin-containing culture solution, and the other group was added with cisplatin-free culture solution. After 12 hours of culture, cells in the cisplatin-containing group were re-introduced at 5×10 ⁶ /ml Inoculate, respectively add 400μg/ml NADH and NADH-free culture medium to continue culturing for 48 hours, trypsinize, fix with 0.5% paraformaldehyde, and increase cell membrane permeability with 0.1% Triton- ¹⁰⁰ . Separate 300 μl tubes, wash, centrifuge, discard the supernatant, add anti-cyclin A monoclonal antibody 6E6, anti-cyclin B ₁ monoclonal antibody V152, anti-cyclin D ₁ monoclonal antibody DCS-6, anti-p53 protein monoclonal antibody to each tube Anti-D0-7, anti-c-myc protein monoclonal antibody C-33, anti-bc1-2 protein monoclonal antibody 124, anti-c-erbB-2 protein monoclonal antibody NCL-CB11, anti-c-fos protein monoclonal antibody UB-06341, mixed Mix well, incubate at 37°C for 1 hour, wash and centrifuge, add 50 μl of FITC-labeled anti-mouse secondary antibody or anti-rabbit secondary antibody to each tube, incubate at 37°C for 30 minutes, wash and centrifuge, shake and suspend in PBS, and detect PC12 survival by flow cytometry and fluorescence intensity of apoptotic cells. 4. Propidium iodide (PI) staining

Collect 10 ⁶ PC12 cells treated with different factors, centrifuge, wash with PBS, add ice-cold 70% ethanol to fix, wash with PBS, add PI fluorescent staining solution (50 μg/ml PI, 10 μg/ml RNase) and incubate at 4°C in the dark After 30 minutes, the DNA fluorescence intensity was analyzed by flow cytometry, and a hypodiploid peak appeared in the histogram during cell apoptosis. 5. Transmission electron microscope observation

Treated PC12 cells were cultured according to the above conditions, scraped from the culture bottle with a cell scraper, prepared by agar pre-embedding method, pre-fixed with 2.5% glutaraldehyde, post-fixed with 1% osmic acid, rinsed, dehydrated, permeated and epoxy Resin embedding, ultra-thin section, sodium acetate and lead citrate staining, transmission electron microscope observation and photographing.

Results: The proliferation activity of PC12 cells increased in the NADH group, the apoptosis rate in the NADH group was significantly lower than that in the cisplatin group, and the cisplatin group showed obvious changes in the morphology of apoptosis, and the cells were mainly blocked in the G ₁ phase with cyclins during apoptosis. D ₁ was highly expressed, and the expressions of p53, bc1-2, c-erbB-2, c-myc, cyclin A, and B ₁ decreased by 52.2%, 60.8%, 21.9%, 90.7%, 40.9%, and 58.5% respectively; After NADH treatment for 48 hours, the expression of bc1-2 and cyclin B ₁ increased significantly. Compared with the control group, the expression of p53 protein in the NADH treatment group decreased by 59.6%, and the protein expression of c-myc and c-erbB-2 in the NADH group decreased , c-fos protein expression increased. The reduced coenzyme NADH not only promotes cell proliferation, but also rescues cisplatin-induced apoptotic damage through up-regulation of bc1-2 protein, cyclin D ₁ , c-fos and down-regulation of p53. Example 2: 1. Tunel and Hoechest 33342 double staining to detect cell apoptosis 4% paraformaldehyde fixed the cells for 30 minutes, after washing with PBS, add 0.1% Tritox-100 and 0.1% sodium citrate for permeabilization, then wash with PBS , add Tunel reaction solution containing terminal deoxynucleotidyl transferase and fluorescent labeling solution to incubate in the dark, wash and then add Hoechest 33342 for incubation, wash with PBS solution and observe and analyze under a fluorescent microscope. 2. Detection of cytoplasmic calcium ions, pH, ROS and mitochondrial membrane potential changes by laser confocal microscopy

¹⁰ cells were inoculated on the central cover glass of the petri dish for cultured cells. After the cells adhered to the wall, they were rinsed with PBS buffer, and fluorescent dyes Rhodamin 123, Fluo-3-AM, Snalf-Calcein-1, and H ₂ DCF were added to each dish. The changes of cytoplasmic calcium ion, pH, ROS and mitochondrial membrane potential were detected by laser confocal microscope. 3. Detection of Caspase-3 and Caspase-8

After centrifugation, add ice-cold lysate and incubate on ice for 10 minutes, take the supernatant and add 2× reaction solutions DEVD-pNA and IETD-pNA, then bathe in water at 37°C for 1 hour, and detect with a UV spectrophotometer at 405 nm. 4. Western Blot was used to detect the expression changes of PARP and cytochrome C before and after apoptosis

Add protein treatment extract to the cultured cells, ultrasonically pulverize the sample, bathe in water at 65°C for 15 minutes, transfer to NC membrane after 10% polyacrylamide gel electrophoresis, block with 1% blocking solution, add PARP polyclonal antibody (1 : 1000) and cytochrome C monoclonal antibody (1: 200) were incubated, washed with TBST buffer, incubated with secondary antibody, washed with TBST buffer, and subjected to AP substrate chromogenic reaction. 5. RT-PCR detection of expression changes of P53, Bc1-2, Fas and β-actin before and after apoptosis

Add Tripure extraction reagent and chloroform to culture cells, centrifuge at 12000g for 15 minutes, take colorless supernatant, add isopropanol, centrifuge at 12000g for 10 minutes, take RNA precipitate, wash with 75% alcohol, dry in vacuum, add DEPC water; reverse transcription reaction : RNA 5μl OligdT 1μl DEPC water 6.5μl 5×RT buffer 4μl 10×dNTP 2μl Rnasin 0.5μl M-Mlv 1μl, incubate at 37°C for 1 hour; PCR reaction: cDNA 5μl 10×dNTP 5μl 10×buffer 5μl Tagase 2U each Primer 2μl DEPC water 32μl, put the reaction tube in a PCR machine at 93°C for denaturation for 45 seconds, 53°C for 45 seconds, 72°C for 45 seconds, a total of 35 cycles, and finally 72°C for 300 seconds, each take 8μl of PCR product 1.5% after loading Agarose gel electrophoresis.

Results: Under the fluorescence microscope, the nuclei of living cells showed diffuse and uniform fluorescence. When apoptosis occurred, densely stained and dense granular fluorescence could be seen in the nucleus or cytoplasm. In the low-dose radiotherapy and chemotherapy treatment groups, intracytoplasmic calcium ions, pH, and ROS were significantly increased, and mitochondrial membrane potential was significantly decreased, while in the NADH protection group, intracytoplasmic calcium ions, pH, and ROS were not increased, and mitochondrial membrane potential was not significantly increased. Variety. Caspase-3 and Caspase-8 increased in the early stage of apoptosis in the low-dose radiotherapy and chemotherapy treatment groups, and NADH could block the expression of apoptosis molecules Caspase-3 and Caspase-8, prevent PARP molecules from breaking into active molecules and cytochrome C from mitochondria released into the cytoplasm. Coenzyme NADH can inhibit apoptosis by up-regulating bc1-2 gene expression and down-regulating p53 gene expression. Embodiment 3: 1. The NADH content in the cells was detected by a circular enzyme analysis method. 2. The protective effect of NADH on normal bone marrow stem cells

Bone marrow stem cells were separated by Ficoll and centrifuged at 400×g for 20 minutes. After washing, they were suspended in TC199 culture medium containing 20% autoplasma, adjusted the cell concentration to 10 ⁶ /ml, and incubated with 400 μg/ml NADH at 37°C for 10 hours. Then incubate with different concentrations of chemotherapeutic drugs for 12 hours, inoculate three culture dishes with a diameter of ³⁵ cm at each point; CFU-GM was counted after 10 days of culture, and long-term cultured stem cells were counted according to the method established by Sutherland et al.

Results: In the early stage of apoptosis, when the cells have not yet undergone morphological changes, with the accumulation of DNA fragmentation, the content of NADH in the cells drops sharply. NADH is the coenzyme of many cellular dehydrogenases and is an important reducing energy for cells to carry out all life activities. Its loss is a fatal factor leading to cell death. Exogenous supplementation of NADH increases the growth activity of PC12 cells, and can inhibit DNA cell damage caused by radiotherapy and cisplatin, repair damaged cells and promote cell differentiation. The concentration of NADH in normal tissue cells is significantly higher than that in tumor tissues, and the difference in NADH concentration between normal tissues and tumor tissues can be utilized to protect normal tissue cells from damage by radiotherapy and chemotherapy without affecting the best curative effect of radiotherapy and chemotherapy. Different doses of cisplatin and doxorubicin were used to treat 12 cases of normal bone marrow stem cells in vitro, and the LD ₉₅ of PCM-CFU-GM was measured. It was found that the LD ₉₅ of the NADH-incubated group before treatment was significantly increased, showing the clear protective effect of NADH . Example 4: Detection of the effect of NADH on radiation damage resistance:

Thirty-six male SD rats, weighing 115±10 grams, were randomly divided into 5 groups: control group, 5 Gy single irradiation group, 8 Gy single irradiation group, 4 Gy/time×2 fractionated irradiation group, NADH protection group , 8 in each group. The animals in the control group were reared as usual, and the animals in the experimental group were placed in a plexiglass mouse box of 20cm×20cm×6cm. Whole-body irradiation with 60CO γ-ray was used, the dose rate was 0.9 Gy/min, and the irradiation time was arranged at 5:00 p.m., and it was completed in 2 days. The peripheral blood images of the animals in each group were checked on the 3rd and 7th day after TBI, the liver and kidney function were checked on the 7th day, and the heart, lung, liver, kidney and thoracic bone marrow were collected for routine pathology after the animal was killed by dislocation of the neck Film preparation and histopathological examination.

Using different whole body irradiation doses and fractional irradiation methods, observe the short-term effects in rats to choose a better NADH dosage regimen. The number of peripheral blood white blood cells decreased significantly (86.2%) on the 3rd day after irradiation, while that in the NADH group only decreased by 35.9%; it recovered significantly on the 7th day, and the liver and kidney functions were basically normal. It was observed in this experiment that as the dose increased, the damage to the heart, lung, liver and bone marrow also increased. Compared with single irradiation, fractional irradiation has less damage to the lungs and slightly more damage to the bone marrow. From lung, liver, kidney and bone marrow of thoracic vertebrae, conventional pathological slides and histopathological examination showed that fractional irradiation of NADH group caused less damage to lung and significantly reduced damage to bone marrow. It shows that NADH has a good anti-radiation damage effect.

Claims (5)

1. coenzyme NAD H control histocytochemistry and radiation damage are characterised in that:

(1) this coenzyme can be avoided chemistry and radiation damage damage and cytotoxicity by selective protection normal tissue cell (cells such as liver, kidney, lung and nerve), can be used as the protection and the treatment of biological and chemical weapons and injury by nuclear.

(2) utilize the difference of NADH concentration between normal structure and the tumor tissues, can reach the purpose that normal tissue cell is avoided the chemicotherapy damage and don't influence the chemicotherapy optimum curative effect, be used for tumor patient chemicotherapy toxicity and prevent and treat medicine.

(3) to protect bone marrow three be hematopoietic stem cell to this coenzyme, can clone the stimulating factor use in conjunction with hemopoietic, can hemopoietic can protect hematopoietic stem cell to avoid radiation again and chemotherapeutic toxicity damages, and is the protection factor of bone marrow.

(4) NADH has obvious anti-cell poison and radiation resistance, increases the body cell emergency reaction ability, improves energy metabolism, increases the effect of opposing and DNA plerosis damage, and is significant for control chemistry and injury by nuclear under the modern war condition.

2. according to claim 1 described coenzyme NAD H, it is characterized by the valid density that is dissolved in normal saline is 20～1000ug/ml, and effectively ratio is 20～90% in excipient.

3. according to claim 1 described coenzyme NAD H, it is characterized by the optium concentration that is dissolved in the normal saline is 200～400ug/ml, and the optimal proportion in excipient is 35～45%.

4. according to claim 1 described coenzyme NAD H, it is characterized by the best use of time is preceding 12 to 24 hours capacities of DNA damage to occur to give.

5. described according to claim 1～4, coenzyme NAD H can be used for the protective agent of chemicotherapy, improves chemicotherapy treatment intensity, improves patient's life quality, and can be used for preventing and treating under the modern war condition effective biological warfare agent of chemistry and injury by nuclear.